A newly identified NES sequence present in spastin regulates its subcellular localization and microtubule severing activity.

Spastin, a microtubule-severing AAA ATPase, regulates microtubule dynamics and plays important roles in cell division and neurogenesis. Mutations in the spastin-coding gene SPAST lead to neurodegenerative disorders and cause spastic paraplegia type 4. Spastin has two main isoforms, M1 and M87, that differ only in the presence or absence of 86 N-terminal amino acids and have alternative splicing variants that lack exon4. The N-terminal region of M1 contains a hydrophobic domain, nuclear localization signal (NLS), and nuclear export signal (NES), which partly explains the differences in the two isoforms' localization. However, the mechanisms involved in regulating isoform localization, and the effects of localization on spastin functions are not fully understood. We found endogenous M1 and M87 shuttled between the nucleus and cytoplasm during the cell cycle. We identified a NES (amino acids 195-204) that spans the microtubule-interacting and endosomal-trafficking domain and exon4 region. Furthermore, the NES sequence contains both the coiled-coil and exon4 region of spastin isoforms. Highly conserved leucine 195 in exon3 and the two residues in exon4 are crucial for predicted coiled-coil formation. Mutations in NES or leptomycin B treatment reduced cytoplasmic localization and microtubule fragmentation in M87 rather than in M1. Phosphomimetic mutation of threonine 306 adjacent to the NLS (amino acids 309-312) inhibited nuclear transport of M87. Our results indicate that the newly identified NES in the spastin isoforms containing exon4 regulates the subcellular localization of spastin in coordination with NLS controlled by the phosphorylation state of spastin, and is involved in microtubule severing.

Different impacts on brain function depending on the mode of delivery.

The prevalence of delivery through cesarean-section (C-section) has been increasing worldwide. Although different modes of delivery, such as vaginal birth and C-section, are associated with incidence of some diseases in humans, little is known about how delivery stimuli affect short- and long-term brain function. Phenotypic analyses of Atp1a2 homozygous knockout (Atp1a2-/-) neonates showed that the mode of delivery affected neural phenotypes; Atp1a2-/- mice born by vaginal delivery started spontaneous breathing, while Atp1a2-/- mice born by C-section showed a complete absence of breathing followed by their death. This life or death phenotype prompted us to examine several aspects of the neonatal brain following C-section or vaginal delivery. We found significantly different levels of several monoamines and transporters/channel proteins and a different c-Fos expression pattern. Furthermore, these mice showed different behaviors in adulthood. Our results suggest that birth mode impacts neurotransmission and functional network formation in the neonatal brain.

Synergistic Antiproliferative Effects of Zoledronic Acid and Fluvastatin on Human Pancreatic Cancer Cell Lines: An in Vitro Study.

Bisphosphonates and statins are known to have antitumor activities against different types of cancer cell lines. In the present study, we investigated the antiproliferative effects of the combination of zoledronic acid (ZOL), a bisphophosphonate, and fluvastatin (FLU), a statin, in vitro on two types of human pancreatic cancer cell lines, Mia PaCa-2 and Suit-2. The pancreatic cancer cell lines were treated with ZOL and FLU both individually and in combination to evaluate their antiproliferative effects using WST-8 cell proliferation assay. In this study, we demonstrated a potent synergistic antiproliferative effect of both drugs when used in combination in both cell lines. Moreover, we studied the molecular mechanism behind this synergistic effect, which was inhibited by the addition of the mevalonate pathway products, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). Furthermore, we aimed to determine the effect of ZOL and FLU combination on RhoA and Ras guanosine 5'-triphosphate (GTP)-proteins. The combination induced a marked accumulation in RhoA and unprenylated Ras. GGPP and FPP reversed the increase in the amount of both proteins. These results indicated that the combination treatment impaired RhoA and Ras signaling pathway by the inhibition of geranylgeranylation and/or farnesylation. This study provides a potentially effective approach for the treatment of pancreatic cancer using a combination treatment of ZOL and FLU.

The unstable CCTG repeat responsible for myotonic dystrophy type 2 originates from an AluSx element insertion into an early primate genome.

Myotonic dystrophy type 2 (DM2) is a subtype of the myotonic dystrophies, caused by expansion of a tetranucleotide CCTG repeat in intron 1 of the zinc finger protein 9 (ZNF9) gene. The expansions are extremely unstable and variable, ranging from 75-11,000 CCTG repeats. This unprecedented repeat size and somatic heterogeneity make molecular diagnosis of DM2 difficult, and yield variable clinical phenotypes. To better understand the mutational origin and instability of the ZNF9 CCTG repeat, we analyzed the repeat configuration and flanking regions in 26 primate species. The 3'-end of an AluSx element, flanked by target site duplications (5'-ACTRCCAR-3'or 5'-ACTRCCARTTA-3'), followed the CCTG repeat, suggesting that the repeat was originally derived from the Alu element insertion. In addition, our results revealed lineage-specific repetitive motifs: pyrimidine (CT)-rich repeat motifs in New World monkeys, dinucleotide (TG) repeat motifs in Old World monkeys and gibbons, and dinucleotide (TG) and tetranucleotide (TCTG and/or CCTG) repeat motifs in great apes and humans. Moreover, these di- and tetra-nucleotide repeat motifs arose from the poly (A) tail of the AluSx element, and evolved into unstable CCTG repeats during primate evolution. Alu elements are known to be the source of microsatellite repeats responsible for two other repeat expansion disorders: Friedreich ataxia and spinocerebellar ataxia type 10. Taken together, these findings raise questions as to the mechanism(s) by which Alu-mediated repeats developed into the large, extremely unstable expansions common to these three disorders.

Anti-inflammatory effect of amlodipine plus atorvastatin treatment on carotid atherosclerosis in zucker metabolic syndrome rats.

To investigate the effects of amlodipine in combination with atorvastatin on carotid atherosclerotic changes in metabolic syndrome, 8-week-old Zucker fatty rats were treated with vehicle, amlodipine, atorvastatin, or amlodipine in combination with atorvastatin for 28 days. Histological studies of common carotid arteries showed that lipid deposition determined by Sudan III staining was significantly reduced in rats treated with amlodipine or atorvastatin alone and was further reduced by amlodipine in combination with atorvastatin. Immunohistochemical studies of the pro-inflammatory cytokine tumor necrosis factor (TNF)-α, the arterial calcification initiator bone morphogenetic protein (BMP) 2, the angiogenic factor Notch1, and the smooth muscle cell marker α-smooth muscle actin (SMA) showed that the high expression of all four protein in vehicle-treated rats was greatly decreased by amlodipine, atorvastatin, or amlodipine in combination with atorvastatin, in ascending order. Double immunostaining showed marked colocalization of TNF-α with bone morphogenetic protein 2 and Notch1 with α-SMA in the vehicle group, which was greatly reduced by amlodipine plus atorvastatin. These data suggest that combination therapy may be more effective in preventing atherosclerotic processes and subsequent carotid vascular events than administrating amlodipine or atorvastatin alone in metabolic syndrome.

Strong neurogenesis, angiogenesis, synaptogenesis, and antifibrosis of hepatocyte growth factor in rats brain after transient middle cerebral artery occlusion.

Hepatocyte growth factor (HGF) and glial cell line-derived neurotrophic factor (GDNF) are strong neurotrophic factors. However, their potentials in neurogenesis, angiogenesis, synaptogenesis, and antifibrosis have not been compared. Therefore, we investigated these effects of HGF and GDNF in cerebral ischemia in the rat. Wistar rats were subjected to 90 min of transient middle cerebral artery occlusion (tMCAO). Immediately after reperfusion, HGF or GDNF was given by topical application. BrdU was injected intraperitoneally twice daily 1, 2, and 3 days after tMCAO. On 14 day, we histologically evaluated infarct volume, antiapoptotic effect, neurogenesis, angiogenesis, synaptogenesis, and antifibrosis. Both HGF and GDNF significantly reduced infarct size and the number of TUNEL-positive cells, but only HGF significantly increased the number of BrdU-positive cells in the subventricular zone, and 5'-bromo-2'-deoxyuridine -positive cells differentiated into mature neurons on the ischemic side. Enhancement of angiogenesis and synaptogenesis at the ischemic boundary zone was also observed only in HGF-treated rats. HGF significantly decreased the glial scar formation and scar thickness of the brain pia mater after tMCAO, but GDNF did not. Our study shows that both HGF and GDNF had significant neurotrophic effects, but only HGF can promote the neurogenesis, angiogenesis, and synaptogenesis and inhibit fibrotic change in brains after tMCAO.

Synergistic benefit of combined amlodipine plus atorvastatin on neuronal damage after stroke in Zucker metabolic rat.

Stroke is a major neurologic disorder and a leading cause of death in the world. We compared neuroprotective effects of single or combination therapy of amlodipine (AM) and atorvastatin (AT) in such a metabolic syndrome model Zucker rat after 90 min of transient middle cerebral artery occlusion (tMCAO). The animals were pretreated with vehicle, AM, AT, or the combination of AM plus AT for 28 days, and at 24h of tMCAO, infarct volume and immunohistochemical analyses were performed. The combination of AM plus AT treatment decreased the infarct volume stronger than each single treatment with AM or AT. The numbers of positive cells of oxidative stress markers such as 8-hydroxy-2'-deoxyguanosin (8-OHdG), 4-hydroxy-2-nonenal (4-HNE), and advanced end glycation products (AGE) and inflammation markers such as tumor necrosis factor alpha (TNF-α) and monocyte chemoattractant protein-1(MCP-1) decreased dramatically in the combination-treated group compared with single AM- or AT-treated group. The present study showed that single AM or AT treatment showed neuroprotective effects both with antioxidative and anti-inflammatory mechanisms, but combination therapy of AM plus AT presented a further synergistic benefit in acute ischemic neural damages.

Temporal and spatial differences of multiple protein expression in the ischemic penumbra after transient MCAO in rats.

Temporal and spatial differences and relationships of proteins relating to the ischemic penumbra were examined at 1, 3, 12, 24, and 48 h after 90 min of transient middle cerebral artery occlusion (tMCAO) in rats. 2, 3, 5-triphenyltetrazolium chloride (TTC) staining showed that the apparent infarction focus first appeared at 1h after tMCAO, which then largely matured at 24h. Immunohistochemistry and Western blot indicated no or trace levels of c-fos, hypoxia inducible factor-1 alpha (HIF-1 alpha), heat shock protein 70 (HSP70), and annexin V (A5) positive cells in the sham control brain. Expression of c-fos increased quickly and widely within and outside of the affected arterial territory (peak at 1h), and that of HIF-1 alpha reached the maximum at 12h in a smaller area than c-fos. HSP70 began to be induced during the first few hours after tMCAO, peaked at 24h, then decreased within 48 h, while A5 was slightly expressed at 3h, then gradually increased until 48 h. Double immunofluorescent analyses showed that the colocalization rates of c-fos/HIF-1 alpha, HIF-1 alpha/HSP70, HSP70/A5, and A5/TUNEL were 40.6%, 58.4%, 42.1% and 61.0%, respectively. These data suggest that multiple molecular penumbra exist after 90 min of tMCAO in the rat brain where several different proteins participate in different temporal and spatial expression patterns. Thus, there is a window for rescue of ischemic neural cells from 12 to 48 h after injury.

Tridermal tumorigenesis of induced pluripotent stem cells transplanted in ischemic brain.

Stroke is a major neurologic disorder. Induced pluripotent stem (iPS) cells can be produced from basically any part of patients, with high reproduction ability and pluripotency to differentiate into various types of cells, suggesting that iPS cells can provide a hopeful therapy for cell transplantation. However, transplantation of iPS cells into ischemic brain has not been reported. In this study, we showed that the iPS cells fate in a mouse model of transient middle cerebral artery occlusion (MCAO). Undifferentiated iPS cells (5 x 10(5)) were transplanted into ipsilateral striatum and cortex at 24 h after 30 mins of transient MCAO. Behavioral and histologic analyses were performed at 28 day after the cell transplantation. To our surprise, the transplanted iPS cells expanded and formed much larger tumors in mice postischemic brain than in sham-operated brain. The clinical recovery of the MCAO+iPS group was delayed as compared with the MCAO+PBS (phosphate-buffered saline) group. iPS cells formed tridermal teratoma, but could supply a great number of Dcx-positive neuroblasts and a few mature neurons in the ischemic lesion. iPS cells have a promising potential to provide neural cells after ischemic brain injury, if tumorigenesis is properly controlled.

Induction of parkinsonism-related proteins in the spinal motor neurons of transgenic mouse carrying a mutant SOD1 gene.

Amyotrophic lateral sclerosis is a progressive and fatal disease caused by selective death of motor neurons, and a number of these patients carry mutations in the superoxide dismutase 1 (SOD1) gene involved in ameliorating oxidative stress. Recent studies indicate that oxidative stress and disruption of mitochondrial homeostasis is a common mechanism for motor neuron degeneration in amyotrophic lateral sclerosis and the loss of midbrain dopamine neurons in Parkinson's disease. Therefore, the present study investigated the presence and alterations of familial Parkinson's disease-related proteins, PINK1 and DJ-1, in spinal motor neurons of G93ASOD1 transgenic mouse model of amyotrophic lateral sclerosis. Following onset of disease, PINK1 and DJ-1 protein expression increased in the spinal motor neurons. The activated form of p53 also increased and translocated to the nuclei of spinal motor neurons, followed by increased expression of p53-activated gene 608 (PAG608). This is the first report demonstrating that increased expression of PAG608 correlates with activation of phosphorylated p53 in spinal motor neurons of an amyotrophic lateral sclerosis model. These results provide further evidence of the profound correlations between spinal motor neurons of amyotrophic lateral sclerosis and parkinsonism-related proteins.

Tannic acid facilitates expression of the polypyrimidine tract binding protein and alleviates deleterious inclusion of CHRNA1 exon P3A due to an hnRNP H-disrupting mutation in congenital myasthenic syndrome.

We recently reported that the intronic splice-site mutation IVS3-8G>A of CHRNA1 that encodes the muscle nicotinic acetylcholine receptor alpha subunit disrupts binding of a splicing repressor, hnRNP H. This, in turn, results in exclusive inclusion of the downstream exon P3A. The P3A(+) transcript encodes a non-functional alpha subunit that comprises 50% of the transcripts in normal human skeletal muscle, but its functional significance remains undetermined. In an effort to search for a potential therapy, we screened off-label effects of 960 bioactive chemical compounds and found that tannic acid ameliorates the aberrant splicing due to IVS3-8G>A but without altering the expression of hnRNP H. Therefore, we searched for another splicing trans-factor. We found that the polypyrimidine tract binding protein (PTB) binds close to the 3' end of CHRNA1 intron 3, that PTB induces skipping of exon P3A and that tannic acid increases the expression of PTB in a dose-dependent manner. Deletion assays of the PTB promoter region revealed that the tannic acid-responsive element is between positions -232 and -74 from the translation initiation site. These observations open the door to the discovery of novel therapies based on PTB overexpression and to detecting possible untoward effects of the overexpression.

Human branch point consensus sequence is yUnAy.

Yeast carries a strictly conserved branch point sequence (BPS) of UACUAAC, whereas the human BPS is degenerative and is less well characterized. The human consensus BPS has never been extensively explored in vitro to date. Here, we sequenced 367 clones of lariat RT-PCR products arising from 52 introns of 20 human housekeeping genes. Among the 367 clones, a misincorporated nucleotide at the branch point was observed in 181 clones, for which we can precisely pinpoint the branch point. The branch points were comprised of 92.3% A, 3.3% C, 1.7% G and 2.8% U. Our analysis revealed that the human consensus BPS is simply yUnAy, where the underlined is the branch point at position zero and the lowercase pyrimidines ('y') are not as well conserved as the uppercase U and A. We found that the branch points are located 21-34 nucleotides upstream of the 3' end of an intron in 83% clones. We also found that the polypyrimidine tract spans 4-24 nucleotides downstream of the branch point. Our analysis demonstrates that the human BPSs are more degenerative than we have expected and that the human BPSs are likely to be recognized in combination with the polypyrimidine tract and/or the other splicing cis-elements.

[Molecular and genetic analysis of spinocerebellar ataxia type 10 (SCA10)].

Spinocerebellar ataxia type 10 (SCA10) is a dominantly inherited neurodegenerative disease caused by expansion of the ATTCT pentanucleotide repeat in intron 9 of a novel gene, ATXN10, on chromosome 22q13.3. It is clinically characterized by progressive ataxia, seizures, and anticipation, which can vary within and between families. The length of the expanded ATTCT repeats is highly unstable on paternal transmission and shows a variable degree of somatic and germline instabilty, revealing complex SCA10 genetic mechanisms. How this untranslated ATTCT expansion leads to neurodegeneration has been still controversial. Growing number of evidence indicates a gain-of-function RNA mechanism, similar to the myotonic dystrophies caused by non-coding CTG or CCTG repeat expansions.

Somatic and germline instability of the ATTCT repeat in spinocerebellar ataxia type 10.

Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant disorder characterized by ataxia, seizures, and anticipation. It is caused by an expanded ATTCT pentanucleotide repeat in intron 9 of a novel gene, designated "SCA10." The ATTCT expansion in SCA10 represents a novel class of microsatellite repeat and is one of the largest found to cause human diseases. The expanded ATTCT repeat is unstably transmitted from generation to generation, and an inverse correlation has been observed between size of repeat and age at onset. In this multifamily study, we investigated the intergenerational instability, somatic and germline mosaicism, and age-dependent repeat-size changes of the expanded ATTCT repeat. Our results showed that (1) the expanded ATTCT repeats are highly unstable when paternally transmitted, whereas maternal transmission resulted in significantly smaller changes in repeat size; (2) blood leukocytes, lymphoblastoid cells, buccal cells, and sperm have a variable degree of mosaicism in ATTCT expansion; (3) the length of the expanded repeat was not observed to change in individuals over a 5-year period; and (4) clinically determined anticipation is sometimes associated with intergenerational contraction rather than expansion of the ATTCT repeat.

Dominantly inherited ataxia and dysphonia with dentate calcification: spinocerebellar ataxia type 20.

We describe a pedigree of Anglo-Celtic origin with a phenotypically unique form of dominantly inherited spinocerebellar ataxia (SCA) in 14 personally examined affected members. A remarkable observation is dentate nucleus calcification, producing a low signal on MRI sequences. Unusually for an SCA, dysarthria is typically the initial manifestation. Mild pyramidal signs and hypermetric saccades are noted in some. Its distinguishing clinical features, each present in a majority of affected persons, are palatal tremor, and a form of dysphonia resembling spasmodic dysphonia. Repeat expansion detection failed to identify either CAG/CTG or ATTCT/AGAAT repeat expansions segregating with the disease in this family. The testable SCA mutations have been excluded. On linkage analysis, the locus maps to chromosome 11, which rules out all the remaining mapped SCAs except for SCA5. While locus homogeneity with SCA5 is not formally excluded, we consider it rather unlikely on phenotypic grounds, and propose that this condition may represent an addition to the group of neurogenetic disorders subsumed under the rubric SCA. The International Nomenclature Committee has made a provisional assignment of 'SCA20', although firm designation will have to await a definite molecular distinction from SCA5.

Unpaired structures in SCA10 (ATTCT)n.(AGAAT)n repeats.

A number of human hereditary diseases have been associated with the instability of DNA repeats in the genome. Recently, spinocerebellar ataxia type 10 has been associated with expansion of the pentanucleotide repeat (ATTCT)(n).(AGAAT)(n) from a normal range of ten to 22 to as many as 4500 copies. The structural properties of this repeat cloned in circular plasmids were studied by a variety of methods. Two-dimensional gel electrophoresis and atomic force microscopy detected local DNA unpairing in supercoiled plasmids. Chemical probing analysis indicated that, at moderate superhelical densities, the (ATTCT)(n).(AGAAT)(n) repeat forms an unpaired region, which further extends into adjacent A+T-rich flanking sequences at higher superhelical densities. The superhelical energy required to initiate duplex unpairing is essentially length-independent from eight to 46 repeats. In plasmids containing five repeats, minimal unpairing of (ATTCT)(5).(AGAAT)(5) occurred while 2D gel analysis and chemical probing indicate greater unpairing in A+T-rich sequences in other regions of the plasmid. The observed experimental results are consistent with a statistical mechanical, computational analysis of these supercoiled plasmids. For plasmids containing 29 repeats, which is just above the normal human size range, flanked by an A+T-rich sequence, atomic force microscopy detected the formation of a locally condensed structure at high superhelical densities. However, even at high superhelical densities, DNA strands within the presumably compact A+T-rich region were accessible to small chemicals and oligonucleotide hybridization. Thus, DNA strands in this "collapsed structure" remain unpaired and accessible for interaction with other molecules. The unpaired DNA structure functioned as an aberrant replication origin, in that it supported complete plasmid replication in a HeLa cell extract. A model is proposed in which unscheduled or aberrant DNA replication is a critical step in the expansion mutation.